The major objectives of this proposal are: 1. To investigate whether the crystalline isozymes of aspartate transaminase (GAT), mitochondrial (M) and supernatant (S), retain the substrate binding and half reaction characteristics of the enzymes in solution. 2. To find out if crystallization affects the reactivity (qualitatively and quantitatively) of strategic residues in the protein. 3. To develop methodology for the assignment of pK values for specific residues in the pyridoxal and pyridoxamine forms of free enzyme and in enzyme-substrate complexes. 4. Attempt to assign a role for the active site lys-258 in each isozyme in enzymatic transamination. 5. To search for the role of dimerization in enzyme function and the properties of the phosphate site in M-GAT. 6. To clarify the stereochemical arrangement of substrates in enzyme substrate complexes in each isozyme, and side of exposure to water soluble chemicals. 7. To measure thermodynamic parameters influencing holoenzyme stability with respect to apoenzyme and from one isozyme with respect to the other. 8. To study the mechanism of weak collisions between M-GAT and glutamate dehydrogenase and study the properties of the possible contact sites. 9. To investigate the biological origin of the multiple forms within isozymes and the isolation of a mammalian factor involved in this process. 10. Use a newly developed, simple quantitative assay to determine the clinical significance of isozyme ratio variations in several human diseases.